The present invention relates to simplified synthesis, carbohydrate-based products and practical use of different carbohydrate-based products. Examples hereof are (Galxcex11-3Gal), GlcNAcxcex21-3Gal, xcex1- or xcex2-glycosides thereof, Galxcex11 -3Gal-containing tri- or higher oligosaccharides, xcex1- or xcex2-glycosides thereof, GlcNAcxcex21-3Gal containing tri-, tetra- or higher oligosaccharides, and derivatives and/or xcex1- or xcex2-glycosides of any of these, Galxcex1-1-3GalGlcNAcxcex21-3Gal, xcex1- or xcex2-glycosides thereof, Galxcex11-3Galxcex21-4GlcNAcxcex21-3Galxcex21-4Glc or other higher oligosaccharides containing the Galxcex11-3Gal-structure, xcex1- or xcex2-glycosides thereof, modified carbohydrates, di-, tri-, oligo- or polyfunctional products containing carbohydrate structures, and the use of the products for synthesis, affinity purification, diagnostic applications and therapy.
During the last years, there has been a great interest in synthesis and application of so-called biologically active carbohydrates, which are present in humans, animals and plants especially in the form of glycoproteins, glycolipids, proteoglycans and glycoaminoglycans, within for example diagnostics and therapeutics. Examples of these carbohydrates are blood group related substances, for instance substances of the type blood group A and blood group B determinants, with the structures GalNAcxcex11-3(Fucxcex11-2)Galxcex2-, and Galxcex11-3(Fucxcex11-2)Galxcex2-, respectively. During the last years there has been a great interest in, for example, carbohydrate structures containing Galxcex11-3Galxcex2- since this type of structures has turned out to be of great importance in the binding of patogens causing infectious diseases in cattle (e.g. diarrhea), and as antigenic structures, e.g. different cancer cells express this epitope, and the Galxcex11-3Galxcex21-4GlcNAc-epitope is of interest for vaccination against different types of cancer.
Also pig organs express the Galxcex11-3Galxcex2-epitope and longer carbohydrate structures containing this structure. Therefore, during the last few years there has been a considerable interest in potential xenotransplantations in connection with the use of carbohydrate structures which contain, for example, Galxcex11-3Galxcex2- and oligosaccharides which contain this structure, for example Galxcex11-3Galxcex21-4GlcNAc and Galxcex11-3Galxcex21-4GlcNAcxcex21-3Galxcex21-4Glc. This type of structures seems to be responsible for the primary antibody-mediated rejection response (hyperacute rejection; HAR) of transplanted pig organs to other species including humans.
The same problem will arise in a human- to -human transplantation if the organ donor has a different blood group than the recipient. This limits the availability of suitable donor organs, especially for patients in acute conditions.
In addition to the structures mentioned above, GlcNAcxcex21-3Gal and tri-, tetra- or higher oligosaccharides which contain this structure, such as e.g. Galxcex21-3GlcNAcxcex21-3Galxcex21-4Glc, are of interest as potential alternatives to antibiotics, because it is known that e.g. pathogenic respiratory bacteria bind to the respiratory tract via such carbohydrate structures. The possibility of inhibiting infections with such structures, or with derivatives thereof, is of considerable interest.
In addition to the therapeutic application there is a lot of interest in e.g. the above mentioned structures for the isolation or removal of cells, enzymes and proteins, employing e.g. affinity chromatography, where the structures are coupled to a separation material, such as agarose. It is very interesting to use such separation materials for isolation of proteins which have specificity towards the structures, e.g., glycosyltransferases (which use the saccharide structures as acceptor substrates), lectins, antibodies, and other proteins.
Also, the application of the structures as diagnostic reagents for determination of a cell (for example a pathogenic bacteria), an enzyme, an antibody, another carbohydrate-bound protein, is of considerable interest.
One example is the application in biosensors (e.g. BiaCore, Pharmacia). Here the carbohydrate structure can be bound to a surface, and the subsequent binding of a cell, an enzyme or a protein to the surface (with bound carbohydrate), results in an electric signal which is related to the degree of binding. As a result, both qualitative and quantitative measurements can be done. Another application is in ELISA (enzyme-linked immunosorbent assay). Here the carbohydrate structure can, e.g., be bound to microtiter wells on an ELISA-plate (e.g. via direct covalent binding to activated wells, for example from NUNC, Danmark), via a spacer molecule, or via conjugation to e.g. bovine serum albumin, which then can be bound to the microtiter wells. A sandwich-ELISA of the standard type can, for instance, then be carried out with such ELISA-plates, where the number of carbohydrate-specific cells, enzymes or proteins in a sample can be quantified with any of the standard methods which are used in ELISA (e.g. application of samples to carbohydrate-modified microtiter plates, followed by washing, application of enzyme-conjugate, e.g. peroxidase conjugated with carbohydrate, or antibody specific to the carbohydrate, followed by washing, addition of enzyme-conjugate and substrate, followed by measurement of the absorbance in the mikrotiter wells.
The main problem in the application of carbohydrates today, for example, in diagnostic applications, in down stream processing, for separation of cells, or proteins, or enzymes, in the vaccination against cancer, for specific removal of (xeno)antibodies from plasma in xenotransplantation employing relevant carbohydrate determinants as affinity ligands, and/or for neutralisation of the antibodies via addition of soluble carbohydrates or derivatives thereof, and for application in the prevention of infectious diseases, is to produce the adequate carbohydrate structures or derivatives thereof in pure form and in sufficient quantities. It is desirable to minimise the use of chemical methods, because they are cumbersome (e.g. multi-step synthesis) and result in side-reactions, resulting in the formation of for example mixtures of stereoisomers (xcex1/xcex2-mixtures). Thus, it would be very attractive to find a suitable method for the synthesis of the xcex11-3-linkage and the xcex21-3- linkage in the above mentioned structures.